PROJECT SUMMARY To advance microbiome-brain studies, the general goals of this collaborative proposal are to provide services to research community by engineering probiotics (e.g., E. coli Nissle 1917 (EcN), Bifidobacteria longum, Lactobacillus plantarum) to biosynthesize various naturally occurring chemicals as prebiotic-probiotic conjugates, which will be marketed as important laboratory agents to study the effects of natural products on neurological diseases by modulating gut microbiome. As a proof of concept, EcN that produces high levels of 5-hydroxytryptophan (5-HTP; a natural biosynthetic precursor of serotonin) will be fed to mice to evaluate its bioavailability, pharmacokinetic, tolerability, modulation of microbiome and toxicity. We have recently developed a novel microbial approach for 5-HTP production by rationally engineering a phenylalanine 4- hydroxylase into a tryptophan 5-hydroxylase and reconstituting a novel 5-HTP biosynthetic mechanism in a model microorganism E.coli (K-12 derivative). The proposed research in phase I will focus on (Aim 1) testing whether the novel microbial approach for producing high level 5-HTP in the E. coli strain BW25113 can be utilized in the probiotic strain EcN (5-HTP+EcN) and (Aim 2) on testing that the 5-HTP+EcN has no adverse effects, and the produced 5-HTP is biologically available. We choose EcN as the host strain because its safety has been well documented, and it can directly interact with mucosal dendritic cells in the gut and promote the absorbance of biomolecules biosynthesized by itself. Furthermore, it can reside in the gut for weeks and realize controlled release and sustained delivery of biosynthesized compounds. HGG Research LLC is a company that will develop methods to synthesize various natural compounds in probiotic strains. Research by the academic partner, currently at the University of Georgia, will evaluate the bioavailability, modulation of microbiome and toxicity. The design of studies by expressing various phytochemicals in different probiotic strains using an inducer-free expression system in a mouse model would be the next stage following this STTR project. Biosynthesis of natural products by probiotics in gut has several advantages compared to oral feeding of these compounds. We believe that the proposed research is innovative and of great significance; and represents a frontier in health research, metabolic engineering and synthetic biology with implications in biology, chemistry, and engineering.